The present invention relates to the detection and quantitation of fetal erythrocytes in adult blood and more particularly to the detection and quantitation of fetal-maternal hemorrhages wherein the mother is Rho(D)-negative and the fetus is Rho(D)-positive.
The detection and quantitation of fetal erythrocytes in adult blood is important in diagnosis of numerous maladies, such as for example, sickle-cell anemia and other hereditary blood disorders. It is also important in the detection of ante-natal placental transfer and fetal-maternal hemorrhage in pregnant women. While the method of the invention is applicable to the detection and quantitation of fetal erythrocytes in adult blood for whatever reason, this latter purpose will be used as an illustration throughout this application.
When there is incompatibility between the blood of a fetus and the blood of the mother, an immunization reaction can occur if there is transfer of fetal blood into the maternal blood stream. Such transfer can occur either through ante-natal placental transfer or through fetal-maternal hemorrhage (FMH) at childbirth, in the process of a spontaneous or induced abortion, or because of obstetric trauma. This immunization has an extremely adverse affect on the ability of the mother to carry a subsequent fetus having the same incompatibility, because the immune reaction built up by the blood of first child may cause spontaneous abortion of the subsequent fetus, hemolytic disease of the newborn, or other disorders. It is therefore desirable in the case of incompatibility, and especially in the case of Rh-incompatibility, to inject the appropriate antibody into the mother just after such transfer has occured in order to react with the fetal antigen and thus prevent the immune reaction from developing.
It is known that approximately 20 micrograms of anti-Rho(D) antibody will suppress the antigenic effect of one ml of packed erythrocytes. It is therefore the procedure at present to administer a dose of approximately 300 micrograms of Rho(D) immune globulin (human) (e.g. RhoGAM globulin manufactured by Ortho Diagnostics, Inc.) to an Rho(D)-negative mother within 72 hours after the delivery of an Rho(D)-positive child, which delivery may involve an FMH. While this dose of RhoGAM globulin is sufficient for neutralizing FMH in the majority of cases, some types of FMH are not neutralized by this dose, probably because of a so-called "massive" hemorrhage (one transferring more than about 15 ml of packed erythrocytes). Further, in the case of ante-natal placental transfer, it is important to monitor the quantities of erythrocytes being transferred. It is therefore necessary for satisfactory treatment of fetal erythrocyte transfer to have a test for determining the extent of said transfer so that the dosage of RhoGAM globulin or other treating agent may be adjusted accordingly.
It is known that fetal hemaglobin is less readily eluted from the fetal erythrocyte than is adult hemoglobin from the adult erythrocyte at a pH of about 3.2-3.3. This fact has been utilized in designing prior art methods to determine the extent of fetal-maternal hemorrhage. In such prior art tests the pH of 3.2-3.3 has been deemed critical to produce optimum differential elution of adult erythrocytes relative to fetal erythrocytes.
The following steps are an outline of prior art methods for detecting fetal erythrocytes in adult blood by differential elution:
A. a smear of blood is prepared on a slide and is dried; PA1 B. the smear is fixed on the slide, for example, by treatment with a loweralkanol, and again air dried; PA1 C. the fixed slide is immersed in an elution buffer to differentially elute the adult erythrocytes relative to the fetal erythrocytes; PA1 D. the eluted smear is stained; and PA1 E. the presence of fetal erythrocytes and number of fetal erythrocytes and adult erythrocytes are determined.
The prior art test which has received the widest clinical acceptance is the so called Kleihauer-Betke test and modifications thereof. In this prior art test, a thin smear of maternal blood which has been treated with an anticoagulent is fixed onto a slide in 80 percent aqueous ethanol. The smear is then eluted in a citric-phosphate buffer at pH 3.3 for five minutes at 37.degree. to cause differential elution of adult erythrocytes relative to fetal erythrocytes, after which the smear is stained with hematoxylin and erythrosin to stain the hemoglobin-containing fetal erythrocytes. Inspection of the slide under a microscope reveals the stained fetal erythrocytes and the colorless, transparent so-called "ghost" eluted adult erythrocytes. In the Kleihauer test and its modifications, the "ghost" erythrocytes are difficult to count both because of their colorless transparency and also because some of the adult erythrocytes are often fragmented during the elution process. There are many variations of this test, but they are all referred to as "Kleihauer tests."
The Kleihauer-Betke test, however, suffers several disadvantages. First, the reagents must be freshly prepared at the time the test is conducted; they are generally not stable for longer than 24 hours. Second, the test takes over an hour to set up and run, excluding the time for the preparation of the reagents. Third, the results are sufficiently variable and sufficiently difficult to interpret that the test is not believed to be truly quantitative. That is, while it is possible using the Kleihauer test to determine the presence of a massive fetal-maternal hemorrhage, it is not always possible to determine the extent of such hemorrhage due to the difficulty in counting the barely-visible "ghost" erythrocytes discussed above.
The method of the present invention obviates these disadvantages of the prior art. It is both rapid and convenient. Moreover, the present method allows ready quantitation of the amount of fetal erythrocytes both because of the visibility of the eluted adult erythrocytes and because there is no fragmentation of adult erythrocytes during the elution. The reagents used therein are stable for long periods of time and may conveniently be stored until needed. Further, these reagents may easily be mass produced.